A High-Throughput Mass-Spectrometry-Based Assay for Identifying the Biochemical Functions of Putative Glycosidases

Abstract

The most commonly employed glycosidase assays rely on bulky ultraviolet or fluorescent tags at the anomeric position in potential carbohydrate substrates, thereby limiting the utility of these assays for broad substrate characterization. Here we report a qualitative mass spectrometry-based glycosidase assay amenable to high-throughput screening for the identification of the biochemical functions of putative glycosidases. The assay utilizes a library of methyl glycosides and is demonstrated on a high-throughput robotic liquid handling system for enzyme substrate screening. Identification of glycosidase biochemical function is achieved through the observation of an appropriate decrease in mass between a potential sugar substrate and its corresponding product by electrospray ionization mass spectrometry (ESI-MS). In addition to screening known glycosidases, the assay was demonstrated to characterize the biochemical function and enzyme substrate competency of the recombinantly expressed product of a putative glycosidase gene from the thermophilic bacterium Thermus thermophilus.

Keywords: assay development; carbohydrates; glycosidases; high-throughput screening; mass spectrometry.

Figure 1

A) ESI-MS spectrum of enzymatic reaction β-galactosidase from E. coli. with 1-methyl-β-galactopyranoside. B) ESI-MS spectrum of control reaction of 1-methyl-β-galactopyranoside (no enzyme added) as their sodiated adducts.

Scheme 1

A) Compounds in the methyl glycoside library, annotated using the Consortium for Functional Glycomics nomenclature. Blue circle: glucose; Yellow circle: galactose; Green circle: mannose; Red triangle: fucose; Orange five-point star: xylose. B) Scheme of glycosidase assay: high throughput screening was set up using Mosquito HTS robotic liquid handing system (TTP Labtech Inc, Cambridge, MA) using a putative glycosidase and the library substrates. Subsequent ESI-MS analysis identifies the product peak (hexose product as scheme illustrates) which allows identification of the enzyme’s function.